Synthesis and antimicrobial activity of novel dicationic &#39;&#39;reversed amidines&#39;&#39;

ABSTRACT

The present invention relates to novel 2,5-bis{[alkyl(or aryl)imino]aminophenyl}furans and thiophenes of the general formula  
                 
 
in which R 1 , R 2 , R 3  and R 4  are each independently selected from the group consisting of H, alkyl, alkoxy, halide, and alkylhalide groups; R 5  is H, alkyl or aryl; R 6  is H, alkyl, aryl, or NR 7 R 8 , in which R 7  and R 8  are each independently selected from the group consisting of H, alkyl and aryl; and X is O, S or NR 9 , in which R 9  is H or alkyl, and to the use of such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 60/246,244 filed Nov. 6, 2000, the disclosure of which isincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersRO1AI 46365-01A2 and RO1GN61587 from the National Institutes of Health.The United States government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the treatment of microbial infections causedby Mycobacterium tuberculosis, Trypanosoma spp., Candida albicans,Aspergillus spp., Cryptosporidium parvum, Giardia lamblia, Plasmodiumspp., Pneumocystis carinii, Toxoplasma gondii, Fusarium solani, andCryptococcus neoformans.

BACKGROUND OF THE INVENTION

The incidence of microbial infections (e.g., mycobacterial, fungal andprotozoal infections) in the immunocompromised population hassignificantly increased over the past several years. In particular,Candida species, especially Candida albicans, are often significantpathogens in patients infected with human immunodeficiency virus (HIV).Another pathogen, Pneumocystis carinii, causes a form of pneumonia (PCP)that is believed to be one of the leading causes of death in patientssuffering from AIDS.

Human African trypanosomiasis (HAT) has reemerged as a threat to over 60million people. Current estimates are that between 350,000 and 450,000people are infected.

Other severe and life-threatening microbial infections are caused byMycobacterium tuberculosis, Aspergillus spp., Cryptosporidium parvum,Giardia lamblia, Plasmodium spp., Toxoplasma gondii, Fusarium solani,and Cryptococcus neoformans.

The antimicrobial properties of dicationic molecules have been studiedsince the 1930's. Compounds of this type have typically utilized amidinegroups as the cationic moieties, and their activities against a numberof pathogens including Cryptosporidium parvum, Giardia lamblia,Leishmania spp., Plasmodium spp., Pneumocystis carinii, Toxoplasmagondii, Trypanosoma spp., Candida albicans, Aspergillus spp., andCryptococcus neoformans have been reported. See e.g., King, H. et al.,Ann. Trop. Med. Parasitol. 1938, 32,177-192; Blagburn, B. L. et al.,Antimicrob. Agents Chemother. 1991, 35, 1520-1523; Bell, C. A. et al.,Antimicrob. Agents Chemother. 1991, 35, 1099-1107; Bell, et al.,Antimicrob. Agents Chemother. 1990, 34, 1381-1386; Kirk, R. et al., Ann.Trop. Med. Parastiol. 1940, 34, 181-197; Fulton, J. D. Ann. Trop. Med.Parasitol. 1940, 34, 53-66; Ivady, V. G. et al., Monatschr.Kinderheilkd. 1958, 106, 10-14; Boykin, D. W. et al., J. Med. Chem.1995, 38, 912-916; Boykin, D. W. et al., J. Med. Chem. 1998, 41,124-129; Francesconi et al., J. Med. Chem. 1999, 42, 2260-2265; Lindsay,D. S. et al., Antimicrob. Agents Chemother. 1991, 35, 1914-1916; Lourie,E. M; et al., Ann. Trop. Med. Parasitol. 1939, 33, 289-304; Lourie, E.M. et al., Ann. Trop. Med. Parasitol. 1939, 33, 305-312; Das, B. P. etal., J. Med. Chem. 1976, 20, 531-536; Del Poeta, M. et al., J.Antimicrob. Chemother. 1999, 44, 223-228; Del Poeta, M. et al.,Antimicrob. Agents Chemother. 1998, 42, 2495-2502; Del Poeta, M. et al.,Antimicrob. Agents Chemother. 1998, 42, 2503-2510.

Despite the broad range of activity exhibited by diamidines, only onecompound of this chemical type, pentamidine, has seen significantclinical use. Pentamidine has been used clinically against Africantrypanosomiasis, antimony-resistant leishmaniasis and P. cariniipneumonia. See e.g., Apted, F. I. C., Pharmacol. Ther. 1980, 11,391-413; Bryceson, A. D. M. et al., Trans. Roy. Soc. Trop. Med. Hyg.1985, 79, 705-714; Hughes, W. T.; et al., Antimicrob. Agents Chemother.1974, 5, 289-293.

A number of compounds in this class of dicationic molecules have beenshown to bind to the minor-groove of DNA at AT-rich sites and thedetails of their interaction with the minor-groove have been elucidatedfrom biophysical studies and from several crystal structures. It ishypothesized that these types of molecules exert their biologicalactivity by first binding to DNA and then by inhibiting one or more ofseveral DNA dependent enzymes (i.e., topoisomerases, nucleases, etc.) orpossibly by direct inhibition of transcription. See, Tanious, F. A. etal., J. Biomol. Struct. & Dyn. 1994, 11, 1063-1083.; Wilson, W. D. etal., J. Am. Chem. Soc. 1998, 120, 10310-10321; Bailly, C. et al.,Anti-Cancer Drug Design, 1999, 14, 47-60; Mazur, et al., J. MolecularBiology 2000, 300, 321-337; Trent, J. O.; et al., J. Med. Chem. 1996,36, 4554-4562; Guerri, A. et al., Nucleic Acids Res. 1998, 26,2873-2878; Laughton, C. A. et al., Biochemistry 1996, 35, 5655-5661;Beerman, T. A. et al., Biochim. Biophys. Acta 1992, 1131, 52-61; Bell,C. A.; et al., Antimicrob. Agents Chemother. 1993, 37, 2668-2673;Dykstra, C. C. et al., Antimicrob. Agents Chemother. 1994, 38,1890-1898; Hildebrandt, E. et al., J. Euk. Microbial. 1998, 45, 112-121;Henderson, D. et al., Nature Medicine 1995, 1, 525-527; Fitzgerald, D.J.; et al., J. Biol. Chem 1999, 274, 27128-27138.

2,5-Diphenylfuran and 2,4-diphenylfuran diamidines have been found to behighly effective treatments in animal models for Pneumocystis cariniiand Cryptosporidium parvum. See Blagburn, B. L. et al., Antimicrob.Agents Chemother. 1991, 35, 1520-1523; Boykin, D. W. et al., J. Med.Chem. 1995, 38, 912-916; Boykin, D. W. et al., J. Med. Chem. 1998, 41,124-129; Francesconi, I. et al., J. Med. Chem. 1999, 42, 2260-2265;Tidwell, R. R. J. Parasitol. 1998, 84, 851-856. Furthermore, thesemolecules have shown antifungal activity in vitro against Candidaalbicans and Cryptococcus neoformans. See, Del Poeta, M. et al., J.Antimicrob. Chemother. 1999, 44, 223-228; Del Poeta, M. et al.,Antimicrob. Agents Chemother. 1998, 42, 2495-2502; Del Poeta, M. et al.,Antimicrob. Agents Chemother. 1998, 42, 2503-2510.

Although there are reports of antimicrobial activity of guanidinocompounds, this class of cationic compounds has not been studied asextensively as their amidino analogs. See Lourie et al., Ann. Trop. Med.Parasitol. 1937, 31, 435-445.

SUMMARY OF THE INVENTION

The synthesis, DNA-binding affinities and antimicrobial properties of2,5-bis{[alkyl(or aryl)imino]aminophenyl}furans and thiophenes aredescribed. These compounds have the imino group of the amidine attachedto an “anilino” nitrogen (in contrast to the known amidino furans inwhich the imino group is directly attached to the aryl ring). Thesecompounds, hereafter, are referred to as “reversed” amidines. Thevarious effects of placing substituents on the central phenyl rings ofthe 2,5-diphenylfuran framework of this class of compounds are alsodescribed.

One aspect of the invention relates to novel compounds that are usefulin treating microbial infections caused by Mycobacterium tuberculosis,Trypanosoma spp., Candida albicans, Aspergillus spp., Cryptosporidiumparvum, Giardia lamblia, Plasmodium spp., Pneumocystis carinii,Toxoplasma gondii, Fusarium solani, and Cryptococcus neoformans.Compounds of the present invention have a structure according to FormulaI:

wherein:

R₁, R₂, R₃ and R₄ are each independently selected from the groupconsisting of H, alkyl, alkoxy, halide, and alkylhalide groups;

R₅ is H, alkyl or aryl;

R₆ is H, alkyl, aryl, or NR₇R₈, wherein R₇ and R₈ are each independentlyselected from the group consisting of H, alkyl and aryl; and

X is O, S or NR₉, wherein R₉ is H or alkyl.

Additional aspects of the invention include pharmaceutical compositionscomprising a compound having a structure according to Formula I, or apharmaceutical salt thereof (i.e., an “active compound”), in apharmaceutically acceptable carrier. Pharmaceutical compositions of thepresent invention are useful in the treatment of microbial infectionscaused by Mycobacterium tuberculosis, Trypanosoma spp., Candidaalbicans, Aspergillus spp., Cryptosporidium parvum, Giardia lamblia,Plasmodium spp., Pneumocystis carinii, Toxoplasma gondii, Fusariumsolani, and Cryptococcus neoformans.

Certain aspects of the invention relate to methods of treating microbialinfections caused by Mycobacterium tuberculosis, Trypanosoma spp.,Candida albicans, Aspergillus spp., Cryptosporidium parvum, Giardialamblia, Plasmodium spp., Pneumocystis carinii, Toxoplasma gondii,Fusarium solani, and Cryptococcus neoformans, in a subject in need ofsuch treatment. The method comprises administering to the subject acompound according to Formulas (I), or a pharmaceutically acceptablesalt thereof, in an amount effective to treat the microbial infection.

A further aspect of the present invention is the use of the activecompounds described herein for the manufacture of a medicament for thetreatment of microbial infections caused by Mycobacterium tuberculosis,Trypanosoma spp., Candida albicans, Aspergillus spp., Cryptosporidiumparvum, Giardia lamblia, Plasmodium spp., Pneumocystis carinii,Toxoplasma gondii, Fusarium solani, and Cryptococcus neoformans in asubject in need of such treatment.

The foregoing and other aspects of the present invention are explainedin detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates various chemical schemes that may be useful in thesynthesis of compounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying specification and drawings, in whichpreferred embodiments of the invention are shown. This invention may,however, be embodied in many different forms and should not be construedas limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

With respect to the compounds of the formulas as used herein, the term“alkyl” refers to C1-10 inclusive, linear, branched, or cyclic,saturated or unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains,including for example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl,pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl,hexynyl, heptynyl, and allenyl groups. The term “alkyl” specificallyincludes cycloakyl hydrocarbon chains, which as used herein refers to C3to C6 cyclic alkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl. In the present invention, preferred alkyls are the loweralkyls. The term “lower alkyl” refers to C1 to C4 linear or branchedalkyl, such as methyl, ethyl, propyl, butyl, isopropyl, sec-butyl, andtert-butyl.

The term “alkyl” also encompasses substituted alkyls, which includeaminoalkyls, hydroalkyls, oxygen-substituted alkyls (i.e., alkoxygroups), and halogen-substituted alkyls (i.e., alkyl halides,polyhaloalkyls). The term “aminoalkyl,” as used herein, refers to C1 toC4 linear or branched amino-substituted alkyl, wherein the term “amino”refers to the group NR′R″, and wherein R′ and R″ are independentlyselected from H or lower alkyl as defined above, i.e., —NH₂, —NHCH₃,—N(CH₃)2, etc. The term “hydroxyalkyl” as used herein refers to C1 to C4linear or branched hydroxy-substituted alkyl, i.e., —CH₂OH, —(CH₂)₂OH,etc. The term “alkoxy” as used herein refers to C1 to C4oxygen-substituted alkyl, i.e., —OCH₃. The term “loweralkoxy,” as usedherein, refers to C1 to C4 linear or branched alkoxy, such as methoxy,ethoxy, propyloxy, butyloxy, isopropyloxy, and t-butyloxy.

The terms “halide” has its conventional meaning and refers to fluoro,chloro, bromo, and iodo groups. Preferred halide groups include chlorogroups, and preferred alkyl halides of the present invention includeCF₃.

The term “aryl” as used herein refers to C3 to C10 cyclic aromaticgroups such as phenyl, naphthyl, and the like, and specifically includessubstituted aryl groups including but not limited to tolyl, substitutedphenyl, and substituted naphthyl. Aryl groups may be substituted withhalide, amino, nitro, and the like. Heterocyclic aromatic rings andpolycyclic aromatic groups are also included in this definition of“aryl.” Specific examples of aryl groups encompassed by the presentinvention include but are not limited to substituted and unsubstitutedpyridine, quinoline, cyclopentadienyl, phenyl, furan, thiophene,pyrrole, pyran, imidazole, isothiazole, isoxazole, pyrazole, pyrazine,pyrimidine, and the like. Preferred aryl groups include pyridine,substituted pyridine (e.g., 5-methylpyridine), and quinoline.

The compounds of the present invention are also useful in the form oftheir pharmaceutically acceptable salt forms. Such salts may include,but are not limited to, the gluconate, lactate, acetate, tartarate,citrate, phosphate, borate, nitrate, sulfate, hydrobromide andhydrochloric salts of the compounds. Compounds of Formula (I) and theirpharmaceutically acceptable salts are referred to herein as “activecompounds” or “active agents.”

The compounds represented by Formula (I) may be formed by synthesisprocedures that are described in the Examples below, as well as bycertain methods known in the art. Some of these known methods are setforth below in the Examples by description or by reference (thedisclosures of which are all incorporated herein by reference in theirentirety).

As noted above, the compounds, methods and compositions of the presentinvention are useful for treating infections caused by Mycobacteriumtuberculosis, Trypanosoma spp., Candida albicans, Aspergillus spp.,Cryptosporidium parvum, Giardia lamblia, Plasmodium spp., Pneumocystiscarinii, Toxoplasma gondii, Fusarium solani, and Cryptococcusneoformans. In a preferred embodiment, the methods and compositions ofthe present invention are used to treat Mycobacterium tuberculosisinfections. In another preferred embodiment, the methods andcompositions of the present invention are used to treat Candida albicansinfections. In another preferred embodiment, the methods andcompositions of the present invention are used to treat infectionscaused by Aspergillus spp. In another preferred embodiment, the methodsand compositions of the present invention are used to treat infectionscaused by Trypanosoma spp. The present invention is useful for treatingall known species of Trypanosoma, with Trypanosoma brucei rhodesienseand Trypanosoma cruzi being particularly preferred. TABLE 1 Examples ofinventive compounds

Compound Number X R₁, R₂ Y 5a O R₁ = R₂ = H NHAm 6a O R₁ = R₂ = HNHC(═NH)-2-Pyr 6b O R₁ = R₂ = H NHC(═NH)Ph 6c O R₁ = R₂ = HNHC(═NH)Ph-4-CH₃ 6d O R₁ = R₂ = H NHC(═NH)-c-hexane 6e O R₁ = R₂ = HNHC(═NH)CH₃ 6f O R₁ = H, R₂ = CH₃ NHC(═NH)CH₃ 5b O R₁ = H, R₂ = CH₃ NHAm5c O R₁ = H, R₂ = OCH₃ NHAm 5d O R₁ = H, R₂ = Cl NHAm 5e O R₁ = H, R₂ =CF₃ NHAm 5f O R₁ = R₂ = CH₃ NHAm 6g O R₁ = H, R₂ = CH₃ NHC(═NH)Ph 6h OR₁ = H, R₂ = CH₃ NHC(═NH)-2-Pyr 6i O R₁ = H, R₂ = CH₃ NHC(═NH)-2-Qu 6j OR₁ = H, R₂ = CH₃ NHC(═NH)-2-Pyr-5-CH₃ 6k O R₁ = H, R₂ = OCH₃NHC(═NH)-2-Pyr 6l O R₁ = H, R₂ = Cl NHC(═NH)-2-Pyr 6m O R₁ = R₂ = CH₃NHC(═NH)-2-Pyr DB 686 S R₁ = R₂ = H NHAm DB 653 S R₁ = R₂ = H NHC(═NH)Ph

Examples of compounds of the present invention are set forth in Table 1,in which Pyr means pyridine, c-hexane means cyclohexane, Ph meansphenyl, Am means amidine, and Qu means quinoline.

In one embodiment of the invention, a subject afflicted with a microbialinfection described herein is administered a therapeutically-effectiveamount of the compound of Formula (I), or a pharmaceutically acceptablesalt thereof. A “therapeutically-effective” amount as used herein is anamount of a compound of Formula (I) that is sufficient to alleviate(e.g., mitigate, decrease, reduce) at least one of the symptomsassociated with the microbial infection. It is not necessary that theadministration of the compound eliminate the symptoms of the infection,as long as the benefits of administration of compound outweigh thedetriments. Likewise, the terms “treat” and “treating”, as used herein,are not intended to mean that the subject is necessarily cured of themicrobial infection, or that all clinical signs thereof are eliminated,only that some alleviation or improvement in the condition of thesubject is effected by administration of the compound of Formula (I).

Suitable subjects of the present invention include humans and animals.When the subject is an animal, mammals are preferred, with livestock andprimates being particularly preferred. Humans are the most preferredsubjects. Subjects may be adult, adolescent, juvenile, infant, orneonatal.

Subjects may be administered the compounds and compositions of thepresent invention by any suitable means. Exemplary means are oraladministration (e.g., in the form of a liquid or solid), intramuscularinjection, subcutaneous injection, and intravenous injection.Pharmaceutical formulations of the present invention comprise activecompounds of the invention in a pharmaceutically acceptable carrier.Suitable pharmaceutical formulations include those suitable forinhalation, oral, rectal, topical, (including buccal, sublingual,dermal, vaginal and intraocular), parenteral (including subcutaneous,intradermal, intramuscular, intravenous and intraarticular) andtransdermal administration. The most suitable route of administration inany given case may depend upon the anatomic location of the conditionbeing treated in the subject, the nature and severity of the conditionbeing treated, and the particular active compound which is being used.The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art.

In the manufacture of a medicament according to the invention (the“formulation”), active compounds or the pharmaceutically acceptablesalts thereof (the “active compounds”) are typically admixed with, interalia, an acceptable carrier. The carrier must, of course, be acceptablein the sense of being compatible with any other ingredients in theformulation and must not be deleterious to the patient. The carrier maybe a solid or a liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a tablet, which maycontain from 0.5% to 99% by weight of the active compound. One or moreactive compounds may be incorporated in the formulations of theinvention, which formulations may be prepared by any of the well knowntechniques of pharmacy consisting essentially of admixing thecomponents, optionally including one or more accessory therapeuticingredients.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, or optionally with one or more accessory ingredients.Compressed tablets may be prepared by compressing, in a suitablemachine, the compound in a free-flowing form, such as a powder orgranules optionally mixed with a binder, lubricant, inert diluent,and/or surface active/dispersing agent(s). Molded tablets may be made bymolding, in a suitable machine, the powdered compound moistened with aninert liquid binder. Formulations for oral administration may optionallyinclude enteric coatings known in the art to prevent degradation of theformulation in the stomach and provide release of the drug in the smallintestine.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound ofFormula (I), or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable, may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound orsalt thereof is an aqueous-soluble salt, using conventional liposometechnology, the same may be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed may be of anyconventional composition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes produced may be reduced in size throughthe use of standard sonication and homogenization techniques or othertechniques known in the art.

Of course, the liposomal formulations containing the pharmaceuticallyactive compounds identified with the methods described herein may belyophilized to produce a lyophilizate which may be reconstituted with apharmaceutically acceptable carrier, such as water, to regenerate aliposomal suspension.

In addition to the active compounds, the pharmaceutical formulations maycontain other additives, such as pH-adjusting additives. In particular,useful pH-adjusting agents include acids, such as hydrochloric acid,bases or buffers, such as sodium lactate, sodium acetate, sodiumphosphate, sodium citrate, sodium borate, or sodium gluconate. Further,the compositions may contain microbial preservatives. Useful microbialpreservatives include methylparaben, propylparaben, and benzyl alcohol.The microbial preservative is typically employed when the formulation isplaced in a vial designed for multidose use.

Pharmaceutical formulations of the present invention may comprisecompounds of the present invention in lyophilized form. Alternatively,pharmaceutical formulations of the present invention may comprisecompounds of the present invention in a pharmaceutically acceptablecarrier. Such pharmaceutical formulations are generally made by admixingthe compounds described herein with a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are preferably liquid,particularly aqueous, carriers, the selection of which are known in theart. For the purpose of preparing such formulations, the compound may bemixed in a buffered saline (e.g., pH 6 to 8) or conventional culturemedia. The formulation may be stored in a sterile glass container sealedwith a rubber stopper through which liquids may be injected andformulation withdrawn by syringe.

With respect to all the methods described herein, a therapeuticallyeffective dosage of any specific compound, the use of which is in thescope of present invention, may vary somewhat from compound to compoundand subject to subject, and will depend upon the condition of thesubject and the route of delivery. A dosage from about 0.5 mg/kg toabout 15 mg/kg of subject body weight, or about 20 mg/kg of subject bodyweight, or even about 25 mg/kg of subject body weight may be employedfor intravenous injection or oral administration.

The concentration of the compound of the present invention or apharmaceutically acceptable salt thereof in a formulation of the presentinvention may be determined by the skilled artisan and will varyaccording to certain conditions, including the characteristics ofsubject being treated (e.g., species, age, weight), the severity andtype of the infecting virus or the strain that the subject is beingvaccinated against, the dosage form being used, and the like.

The compounds of the present invention may be administered inconjunction with other antiviral compounds, as may be determined by theskilled artisan.

The present invention is explained in greater detail in the Exampleswhich follow. These examples are intended as illustrative of theinvention, and are not to be taken as limiting thereof.

EXAMPLE 1 General Methodology: Chemical Synthesis and Analysis

Melting points were determined with a MEL-TEMP® 3.0 capillary meltingpoint apparatus and are uncorrected. ¹H nuclear magnetic resonancespectra were recorded on a Varian Unity+300 or a Varian VRX 400instrument, with peak assignments relative to residual DMSO (2.49 ppm)or CHCl₃ (7.24 ppm). Mass spectra were recorded on a VG Instruments70-SE spectrometer at the Georgia Institute of Technology, Atlanta, Ga.Elemental analyses were performed by Atlantic Microlab, Norcross, Ga.All final compounds were dried in vacuo (oil pump) at 50-60° C. for atleast 36 hours before elemental analysis. Unless otherwise stated, allreagent chemicals and solvents (including anhydrous solvents) werepurchased from Aldrich Chemical Co., Fisher Scientific, or LancasterSynthesis and used as received. Acetonitrile (CaH₂), triethylamine(CaH₂), and ethanol (Mg/I₂) were distilled from the indicated dryingagent. 2,6-Dimethyl-4-nitrobromobenzene andS-(2-Naphthylmethyl)thioacetimidate were prepared according to theliterature. See B. M. Wepster, Rec. Trav. Chim. 73, 809-818 (1954); D.N. Kravtsov, J. Organometal. Chem. 36, 227-237 (1972); B. G. Shearer etal., Tetrahedron Lett. 38, 179-182 (1997).

FIG. 1 sets forth representative schemes for the synthesis of compoundsof the present invention. Although the schemes set forth in the figurerelate to furan compounds, the methods illustrated therein may also becarried out with analogous compounds such as thiophene compounds.Referring to FIG. 1, the synthesis for target compounds required thecorresponding diamino compounds (Scheme 1). The synthesis of the diaminocompounds begins with Stille coupling between a 2,5-distannyl furan orthiophene and substituted bromonitroarenes to form the corresponding2,5-bis[nitrophenyl]furans and thiophenes. Reduction of the2,5-bis-nitrophenyl heterocycles either by catalytic hydrogenation or bystannous chloride produced the desired diamino compounds (Scheme 1). Therequired diamino analogs were obtained by the two step conversion of2,5-bis[nitrophenyl]furans and thiophenes to amines involving Pd(0)coupling of benzophenone imine to form the corresponding aryliminocompounds. The diguanidinium analogs were prepared by the reaction ofthe aryl diamines with Boc-protected S-methylthiourea in the presence ofmercuric chloride (Scheme 2). Reaction of the aryl diamines with twoequivalents of S-(2-naphthylmethyl)thiobenzimidate produced the“reversed” amidines in good yields (Scheme 4).

The 2,5-bis(4-aminophenyl)furans and -thiophenes (Scheme 1) wereprepared in good yield by reduction of the corresponding bis-nitroderivatives using either catalytic hydrogenation (Pd/C), stannouschloride, or iron/AcOH. The bis-nitro derivatives, in turn, wereprepared by the palladium catalyzed coupling of2,5-bis(tri-n-butylstannyl)furan or 2,5-bis(trimethylstannyl)thiophenewith the appropriate halonitrobenzene.

In the following examples, compound numbers refer to the correspondingcompounds in FIG. 1.

EXAMPLE 2 Preparation of 2,5-bis(4-nitrophenyl)furans

The following representative procedures are variations of a generalprocedure previously described in A. Kumar et al., Heterocyclic Comm. 5,301-304 (1999).

2,5-Bis(4-nitrophenyl)furan (Compound 2a). Yield: 88%; orange fluffysolid; mp 269-270° C. (not recrystallized), lit. mp 270-272° C., Ling,C. et al., J. Am. Chem. Soc. 1994, 116, 8784-8792.

2,5-Bis(2-methyl-4-nitrophenyl)furan (Compound 2b). To a solution of2-bromo-5-nitrotoluene (4.32 g, 20 mmol) andtetrakis(triphenylphospine)palladium (0) (0.40 g) in anhydrous1,4-dioxane (50 ml) was added 2,5-bis(tri-n-butylstannyl)furan (6.46 g,10 mmol) and the mixture was heated overnight under nitrogen at 95-100°C. The resulting orange suspension was diluted with hexanes (15 ml),cooled to room-temperature, and filtered to give, after rinsing withhexanes, an orange solid (3.10 g), mp 241-243° C. The product wasrecrystallized from DMF (100 ml) to give a bright orange fluffy solid(2.87 g, 85%), mp 242-243° C. ¹H NMR (DMSO-d₆): 2.69 (s, 6H), 7.31 (s,2H), 8.12 (m, 4H), 8.23 (s, 2H). Anal. Calcd. for C₁₈H₁₄N₂O₅ (338.31):C, H, N.

2,5-Bis(2-methoxy-4-nitrophenyl)furan (Compound 2c). Yield: 77%; brightorange granular solid; mp 308-310° C. (DMF). ¹H NMR (DMSO-d₆): 4.10 (s,6H), 7.37 (s, 2H), 7.90 (s, 2H), 7.94 (d, 2H), 8.22 (d, 2H). Anal.Calcd. for C₁₈H₁₄N₂O₇.0.1H₂O (372.11): C, H, N.

2,5-Bis(2-chloro-4-nitrophenyl)furan (Compound 2d). Yield: 71%; fluffyorange solid; mp 247-247.5° C. (DMF/MeOH). ¹H NMR (DMSO-d₆): 7.70 (s,2H), 8.29 (dd, J=8.8, 2.2 Hz, 2H), 8.36 (d, J=8.8 Hz, 2H), 8.43 (d,J=2.2 Hz, 2H). Anal. Calcd. for C₁₆H₈Cl₂N₂O₅ (379.15): C, H, N.

2,5-Bis(4-nitro-2-trifluoromethylphenyl)furan (Compound 2e). Yield: 74%;fluffy golden needles; mp 158.5-159° C. (EtOH). ¹H NMR (DMSO-d₆): 7.38(s, 2H), 8.24 (d, J=8.7 Hz, 2H), 8.57 (d, J=2.4 Hz, 2H), 8.62 (dd,J=8.6, 2.4 Hz, 2H). Anal. Calcd. for C₁₈H₈F₆N₂O₅ (446.26): C, H, N.

2,5-Bis(2,6-dimethyl-4-nitrophenyl)furan (Compound 2f). Yield: 65%;yellow needles; mp 156.5-157.5° C. (DMF/EtOH/H₂O). ¹H NMR (DMSO-d₆):2.34 (s, 12H), 6.85 (s, 2H), 8.04 (s, 4H). Anal. Calcd. for C₂₀H₁₈N₂O₅(366.36): C, H, N.

EXAMPLE 3 Preparation of 2,5-bis(4-aminophenyl)furans

(The following procedures are representative)

2,5-Bis(4-aminophenyl)furan (Compound 3a). Yield: 94%; pale green/tansolid; mp 218-221° C., lit⁴⁶ mp 213-216° C. MS (El): m/z 250 (M⁺).

2,5-Bis(4-amino-2-methylphenyl)furan (Compound 3b). To a suspension ofthe bis-nitro derivative 2b (2.87 g) in EtOAc (90 ml) and dry EtOH (10ml) was added Pd/C (10%) (0.40 g) and the mixture was hydrogenated on aParr apparatus at an initial pressure of ˜50 psi. After the uptake ofhydrogen subsided (generally 3-6 hours), the resulting solution wasfiltered over Celite and the pale yellow to colorless filtrate wasconcentrated in vacuo to near dryness to give, after dilution withhexanes, the pure diamine as a pale yellow/green solid (2.17 g, 91%), mp174-176° C., which required no purification. ¹H NMR (DMSO-d₆): 2.33 (s,6H), 5.15 (br s, 4H), 6.42 (s, 2H), 6.46 (m, 4H), 7.35 (d, 2H). MS (EI):m/z 278 (M⁺).

2,5-Bis(4-amino-2-methoxyphenyl)furan (Compound 3c). The original oilwas reconcentrated with benzene to give a yellow/tan solid which wastriturated with ether. Yield: 79%; mp 201-202.5° C. ¹H NMR (DMSO-d₆):3.80 (s, 6H), 5.25 (br s, 4H), 6.24 (dd, J=8.3, 2.0 Hz, 2H), 6.30 (d,J=1.9 Hz 2H), 6.56 (s, 2H), 7.48 (d, J=8.4 Hz, 2H). MS (EI): m/z 310(M⁺).

2,5-Bis(4-amino-2-chlorophenyl)furan (Compound 3d). To a suspension ofthe corresponding bis-nitro derivative 2d (1.22 g, 3.2 mmol) in dry EtOH(100 ml) and DMSO (20 ml) was added SnCl₂.2H₂O (5.80 g, 25.7 mmol) andthe mixture was heated under nitrogen at 80° C. After 4-5 hours, TLCshowed that starting material had been consumed, and thus the mixturewas cooled, neutralized with NaOH (aq), and extracted with EtOAc. Theextract was washed with water, brine, then dried (Na₂SO₄) andconcentrated. The resulting oil was crystallized from benzene/hexanewith partial concentration to give a light brown solid (0.74 g, 71%), mp191.5-193° C. Catalytic hydrogenation was not explored. ¹H NMR(DMSO-d₆): 5.60 (br s, 4H), 6.61 (dd, J=8.6, 2.2 Hz, 2H), 6.68 (d, J=2.2Hz 2H), 6.82 (s, 2H), 7.56 (d, J=8.6 Hz, 2H). MS (EI): m/z 318 (M⁺).

2,5-Bis(4-amino-2-trifluoromethylphenyl)furan (Compound 3e). Originalred oil crystallized from EtOAc/hexanes in two crops as a red/orangesolid. Combined yield: 81%; mp (first/major crop) 89.5-91° C.; mp(second crop) 91.5-92° C. ¹H NMR (DMSO-d₆): 5.79 (br s, 4H), 6.52 (s,2H), 6.82 (dd, J=8.4, 2.4 Hz, 2H), 6.98 (d, J=2.2 Hz, 2H), 7.43 (d,J=8.4 Hz, 2H). MS (EI): m/z 386 (M⁺).

2,5-Bis(4-amino-2,6-dimethylphenyl)furan (Compound 3f). Yield: 99%;white fluffy solid; mp 144.5-146° C. ¹H NMR (DMSO-d₆): 2.01 (s, 6H),5.06 (br s, 4H), 6.24 (s 2H), 6.29 (s, 4H). MS (EI): m/z 306 (M⁺).

EXAMPLE 4 Preparation of 2,5-bis(4-N,N′-di-BOC-guanidinophenyl)furanderivatives

(The following procedures are representative) (See Scheme 2).

2,5-Bis(4-N,N′-di-BOCguanidinophenyl)furan (Compound 4a). To aroom-temperature solution of 2,5-bis(4-aminophenl)furan (0.626 g, 2.5mmol) and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (1.56g, 5.3 mmol) in anhydrous DMF was added triethylamine (1.59 g, 15.7mmol) followed by mercury(II) chloride (1.57 g, 5.8 mmol) and theresulting suspension was stirred at room-temperature for 22 hours. Afterdiluting with CH₂Cl₂ and sodium carbonate solution, the suspension wasfiltered over Celite and the filtrate was washed well with water (3×)and finally with brine. After drying (Na₂SO₄), the solvent was removedin vacuo and the residue was diluted with MeOH to give the BOC-protectedbis-guanidine as a pale yellow solid. The collected product was purifiedby reprecipitation from CH₂Cl₂/MeOH to give a fluffy yellow solid (1.25g, 68%), mp>400° C. dec. ¹H NMR (CDCl₃): 1.50 and 1.53 (2s, 36H), 6.65(s, 2H), 7.66 (s, 8H), 10.38 (br s, 2H), 11.61 (br s, 2H).

2,5-Bis(2-methyl-4-N,N′-di-BOCguanidinophenyl)furan (Compound 4b).Yellow solid, mp>250° C. dec. Yield: 62%. ¹H NMR (CDCl₃): 1.51 and 1.52(2s, 36H), 2.53 (s, 6H), 6.60 (s, 2H), 7.40 (s, 2H), 7.62 (d, 2H), 7.74(d, 2H), 10.34 (s, 2H), 11.62 (br s, 2H).

2,5-Bis(2-methoxy-4-N,N′-di-BOCguanidinophenyl)furan (Compound 4c).Yellow solid, mp>300° C. dec. Yield: 79%. ¹H NMR (CDCl₃): 1.50 and 1.53(2s, 36H), 3.95 (s, 6H), 6.95 (s, 2H), 7.13 (d, 2H), 7.59 (s, 2H), 7.86(d, 2H), 10.36 (s, 2H), 11.55 (br s, 2H).

2,5-Bis(2-chloro-4-N,N′-di-BOCguanidinophenyl)furan (Compound 4d). Paleyellow/tan solid, mp>400° C. dec. Yield: 63%. ¹H NMR (CDCl₃): 1.52 (s,36H), 7.17 (s, 2H), 7.63 (dd, 2H), 7.79 (d, 2H), 7.88 (d, 2H), 10.43 (s,2H), 11.59 (br s, 2H).

2,5-Bis(2-trifluoromethyl-4-N,N′-di-BOC guanidinophenyl)furan (Compound4e). Bright orange solid. Yield: 88%. ¹H NMR (CDCl₃): 1.51 and 1.53 (2s,36H), 6.77 (s, 2H), 7.82 (d, 2H), 7.94 (s, 2H), 8.00 (d, 2H), 10.52 (s,2H), 11.59 (br s, 2H).

2,5-Bis(2,6-dimethyl-4-N,N′-di-BOC guanidinophenyl)furan (Compound 4f).Pale yellow/off-white solid, mp>300° C. dec. Yield: 89%. ¹H NMR (CDCl₃):1.51 and 1.53 (2s, 36H), 2.23 (s, 12H), 6.31 (s, 2H), 7.33 (s, 4H),10.27 (s, 2H), 11.63 (br s, 2H).

EXAMPLE 5 Deprotection of N,N′-di-BOC guanidines

(The following procedures are representative).

2,5-Bis(4-guanidinophenyl)furan dihydrochloride (Compound 5a). Asolution of the corresponding N,N′-di-BOCguanidine (1.19 g, 1.62 mmol)in CH₂Cl₂ (15 ml) was diluted with dry EtOH (10 ml) and saturated atice-water bath temperature with anhydrous HCl. The solution was thenstirred at room-temperature for 2-3 days (drying tube), with the productslowly precipitating (shorter reaction times generally gave incompletedeprotection). The resulting suspension was concentrated to neardryness, with the solid then taken up in hot EtOH. After filtering toclarify, the solution was concentrated to near dryness to give asuspension, which was diluted with ether and collected to yield, afterdrying in vacuo at 50-60° C. for 2 days, the bis-guanidinedihydrochloride as an off-white/tan solid (0.66 g, quantitative),mp>300° C. dec. ¹H NMR (DMSO-d₆): 7.12 (s, 2H), 7.31 (d, 4H), 7.58 (brs, 8H), 7.86 (d, 4H), 10.09 (br s, 2H). MS (FAB, thioglycerol): m/z335.3 (MH⁺, 100). Anal. Calcd. for C₁₈H₁₈N₆O.2HCl.0.25EtOH (407.30): C,H, N.

2,5-Bis(4-guanidino-2-methylphenyl)furan dihydrochloride (Compound 5b).Tan solid, mp 265-271° C. dec. ¹H NMR (DMSO-d₆): 2.53 (s, 6H), 6.93 (s,2H), 7.17 (m, 4H), 7.56 (br s, 8H), 7.82 (d, 2H), 10.06 (br s, 2H). MS(FAB, thioglycerol): m/z 363.3 (MH⁺, 100). Anal. Calcd. forC₂₀H₂₂N₆O.2HCl.1.5H₂O.0.66EtOH (496.93): C, H, N.

2,5-Bis(4-guanidino-2-methoxyphenyl)furan dihydrochloride (Compound 5c).Light brown solid. ¹H NMR (DMSO-d₆): 3.95 (s, 6H), 6.92 (dd, 2H), 6.99(d, 2H), 7.02 (s, 2H), 7.58 (br s, 8H), 7.95 (d, 2H), 10.08 (br s, 2H).MS (EI): m/z 352 (M⁺—NH₂CN, 38.0), 310 (100), 267 (38.9), 251 (8.8), 155(18.7). Anal. Calcd. for C₂₀H₂₂N₆O₃.2HCl.1.0H₂O.0.33EtOH (500.57): C, H,N.

2,5-Bis(2-chloro-4-guanidinophenyl)furan dihydrochloride (Compound 5d).Tan solid, mp 300-304° C. dec. ¹H NMR (DMSO-d₆): 7.31 (s, 2H), 7.33 (d,2H), 7.47 (s, 2H), 7.72 (br s, 8H), 8.04 (d, 2H). MS (DCI, ammonia): m/z365, 363, 361 (MH⁺—NH₂CN, 8, 52, 78), 323, 321, 319 (11, 66, 100). Anal.Calcd. for C₁₈H₁₆Cl₂N₆O.2HCl.0.5H₂O (485.21): C, H, N, Cl.

2,5-Bis(4-guanidino-2-trifluoromethylphenyl)furan dihydrochloride(Compound 5e). Orange/red solid. ¹H NMR (DMSO-d₆): 6.99 (s, 2H), 7.63(d, 2H), 7.69 (s, 2H), 7.79 (br s, 8H), 7.91 (d, 2H), 10.37 (br s, 2H).MS (CI, isobutane): m/z 471 (MH⁺, 14), 429 (100), 387 (19). Anal. Calcd.for C₂₀H₁₆F₆N₆O.2HCl.0.67H₂O.0.67EtOH (586.24): C, H, N.

2,5-Bis(4-guanidino-2,6-dimethylphenyl)furan dihydrochloride (Compound5f). Off-white solid. ¹H NMR (DMSO-d₆): 2.20 (s, 12H), 6.56 (s, 2H),7.01 (s, 4H), 7.57 (br s, 8H), 10.09 (br s, 2H). MS (FAB, thioglycerol):m/z 391.2 (MH⁺, 100). Anal. Calcd. for C₂₂H₂₆N₆O.2HCl.0.5H₂O (472.41):C, H, N.

2,5-Bis[4-(benzimidoyl)aminophenyl]thiophene Free base: yellowcrystalline solid, mp 284-286° C. dec (DMF/MeOH/H₂O). Yield: 35%. ¹H NMR(DMSO-d₆): 6.41 (br s, 4NH), 6.91 (d, 4H), 7.40 (s, 2H), 7.44 (d, 6H),7.62 (d, 4H), 7.97 (d, 4H). Hydrochloride: yellow/orange solid, mp304-306° C. dec. ¹H NMR (DMSO-d₆): 7.56 (d, 4H), 7.67 (t, 4H), 7.70 (s,2H), 7.77 (t, 2H), 7.90 (d, 4H), 7.95 (d, 4H), 9.13 (br s, 2H), 9.94 (brs, 2H), 11.71 (br s, 2H). MS (EI): m/z 472 (M⁺, 35.1), 369 (76.8), 266(100), 103 (49.5), 76 (14.8). Anal. Calcd. for C₃₀H₂₄N₄S.2HCl.0.5H₂O(554.52): C, 64.97; H, 4.91; N, 10.10. Found: C, 64.95; H, 4.89; N,10.14.

2,5-Bis[2-methyl-4-(2-pyridylimino)aminophenyl]thiophene Free base:yellow crystals, mp 152-153° C. (EtOH/H₂O). Yield: 20%. ¹H NMR(DMSO-d₆): 2.46 (s, 6H), 6.60 (br s, 4NH), 6.82 (d, 2H), 6.90 (s, 2H),7.16 (s, 2H), 7.42 (d, 2H), 7.55 (m, 2H), 7.95 (t, 2H), 8.30 (d, 2H),8.63 (dd, 2H). Hydrochloride: Yellow powder, mp xxx° C. dec. ¹H NMR(DMSO-d₆): 2.54 (s, 6H), 7.36 (s, 2H), 7.39 (d, 2H), 7.48 (s, 2H), 7.66(d, 2H), 7.85 (m, 2H), 8.22 (t, 2H), 8.47 (d, 2H), 8.89 (d, 2H), 9.37(br s, 2H), 10.12 (br s, 2H), 11.87 (br s, 2H). MS (EI):. Anal. Calcd.for C₃₀H₂₆N₆S.2.5HCl.1.25H₂O (616.30): C, 58.46; H, 5.07; N, 13.64; Cl,14.38. Found: C, 58.83; H, 4.92; N, 13.68; Cl, 14.02.

EXAMPLE 6 Preparation of Compounds 6b-6c (Scheme 3)

The original route to the reversed amidines (which was used to prepare6b-c) is as follows.

2,5-Bis[4-(benzimidoylamino)phenyl]furan Dihydrochloride (Compound 6b)To a chilled solution of 2,5-bis(4-aminophenyl)furan (0.25 g, 1.0 mmol)in dry acetonitrile (10 ml) was added triethylamine (0.22 g, 2.1 mmol)followed dropwise by benzoyl chloride (0.30 g, 2.1 mmol) and theresulting suspension was stirred at room-temperature for 3 hours. Water(10 ml) was then added and the precipitate was collected, rinsed withwater, followed by MeOH, and finally dried in vacuo to give2,5-bis(4-benzamidophenyl)furan as a tan solid (0.44 g, 96%), mp312-314.5° C. ¹H NMR (DMSO-d₆): 6.98 (s, 2H), 7.52-7.62 (m, 6H), 7.80(d, 4H), 7.89 (d, 4H), 7.97 (d, 4H), 10.33 (br s, 2H).

The intermediate bis(benzamide) (0.44 g, 0.96 mmol) was suspended inanhydrous dichloromethane (40 ml) and treated with freshly distilledthionyl chloride (0.68 g, 5.7 mmol) along with 2 drops of DMF and themixture was refluxed with vigorous stirring until a solution wasobtained (20 hours). The solution was then concentrated in vacuo to givea yellow solid, which was co-evaporated with dry benzene. The obtainedimidoyl chloride was dissolved in anhydrous dichloromethane (40 ml) andthe solution was saturated at ice/water-bath temperature with anhydrousammonia and sealed. After stirring overnight at room-temperature, theturbid mixture was concentrated to give a yellow solid, which wastriturated with 0.5N NaOH, collected, and air dried. This free-base(0.44 g, 100%) was dissolved in boiling EtOH (50 ml), filtered, and atice-bath temperature was treated with dry HCl. After failed attempts atinducing precipitation with the addition of ether, the solution wasconcentrated (high vacuum) to give the dihydrochloride as an orangehygroscopic solid, mp 242-248° C. ¹H NMR (DMSO-d₆): 7.26 (s, 2H), 7.58(d, 4H), 7.67 (t, 4H), 7.78 (t, 2H), 7.95 (d, 4H), 8.03 (d, 4H), 9.12(br s, 2H), 9.94 (br s, 2H), 11.66 (br s, 2H). MS (EI): m/z 456 (M⁺,100), 353 (63), 250 (62), 221 (16), 130 (15), 103 (41), 76 (14), 44(22). Anal. Calcd. for C₃₀H₂₄N₄O.2HCl.0.H.0.1 (C₂H₅)(545.87):C, H, N.

2,5-Bis[4-[(4-methylbenzimidoyl)amino]phenyl]furan Dihydrochloride(Compound 6c). Following the above procedure,2,5-bis[(4-methylbenzamido)phenyl]furan was first obtained as a paleyellow solid by reaction of 2,5-bis(4-aminophenyl)furan (0.50 g, 2.0mmol) with 4-methylbenzoyl chloride (0.65 g, 4.2 mmol). Yield: 0.96 g,99%; mp 348-350.5° C. ¹H NMR (DMSO-d₆): 2.39 (s, 6H), 6.98 (s, 2H), 7.34(d, 4H), 7.79 (d, 4H), 7.89 (dd, 8H), 10.25 (br s, 2H).

Subsequent conversion of the bis(benzamide) to the amidine wasaccomplished as above, with the exception that the precipitated productfollowing reaction with ammonia was collected by filtration and rinsedwith EtOH to give the free base directly (Yield: 34%). Thedihydrochloride was obtained as an orange oily solid which crystallizedin vacuo, mp 227-240° C. (hygroscopic). ¹H NMR (DMSO-d₆): 2.44 (s, 6H),7.24 (s, 2H), 7.46 (d, 4H), 7.56 (d, 4H), 7.87 (d, 4H), 8.01 (d, 4H),9.02 (br s, 2H), 9.89 (br s, 2H), 11.66 (br s, 2H). MS (EI): m/z 484(M⁺, 59), 367 (86), 250 (100), 221 (21), 130 (20), 117 (69), 90 (19), 44(36). Anal. Calcd. for C₃₂H₂₈N₄O.2HCl.0.5H₂O (566.51): C, H, N.

EXAMPLE 7 Alternative preparation of bis-{[alkyl(oraryl)imino]aminophenyl}furan derivatives (Scheme 4)

The following experimental is representative. In some cases, the productwas purified by recrystallization.

2,5-Bis[2-methyl-4-(2-pyridylimino)aminophenyl]furan ((Compound 6h). Toa solution of 2,5-bis(4-amino-2-methylphenyl)furan (0.30 g, 1.08 mmol)in dry MeCN (5 ml) was added dry EtOH (15 ml) and the solution waschilled briefly on an ice/water bath.S-(2-Naphthylmethyl)thiobenzimidate hydrobromide (0.815 g, 2.27 mmol)was then added and the mixture was stirred overnight atroom-temperature. The resulting solution was concentrated to an oil,which was triturated with ether to give a yellow solid. The solid wascollected, dissolved in EtOH and basified with NaOH (1N), and the freebase was extracted into EtOAc. After drying (Na₂SO₄) and removing themost of the solvent, the resulting suspension was diluted with excessether to give a fluffy yellow solid (0.36 g, 69%), mp 188-189° C., whichrequired no purification. ¹H NMR (DMSO-d₆): 2.51 (s, 6H), 6.60 (br s,4NH), 6.77 (s, 2H), 6.87 (m, 4H), 7.55 (dd, 2H), 7.74 (d, 2H), 7.95 (m,2H), 8.31 (d, 2H), 8.63 (d, 2H).

To prepare the hydrochloride salt, the free base was suspended in EtOH(40 ml) and treated with dry HCl gas for 5-10 min at ice-bathtemperature. Continued stirring of the resulting solution for 15-20minutes gave an orange suspension which was diluted with ether (40 ml)and filtered to yield an orange powder (0.40 g), mp>180° C. dec. ¹H NMR(DMSO-d₆): 2.62 (s, 6H), 7.08 (s, 2H), 7.44 (d, 2H), 7.47 (s, 2H), 7.85(dd, 2H), 7.99 (d, 2H), 8.22 (t, 2H), 8.49 (d, 2H), 8.89 (d, 2H), 9.36(br s, 2H), 10.13 (br s, 2H), 11.88 (br s, 2H). MS (EI): m/z 486 (M⁺,100), 382 (77.9), 278 (12.8), 104 (20.0), 78 (8.8), 43 (28.9). Anal.Calcd. for C₃₀H₂₆N₆O.3.5HCl.0.5H₂O (623.20): C, H, N, Cl.

2,5-Bis[4-(2-pyridylimino)aminophenyl]furan (Compound 6a) Free base:yellow crystalline solid, mp 221-223° C. (DMF/EtOH/H₂O). Yield: 65% ¹HNMR (DMSO-d₆): 6.80 (br s, 4NH), 6.94 (s, 2H), 7.03 (d, 4H), 7.56 (m,2H), 7.77 (d, 4H), 7.96 (m, 2H), 8.32 (d, 2H), 8.64 (m, 2H).Hydrochloride: Orange/red powder, mp>175° C. dec. ¹H NMR (DMSO-d₆): 7.26(s, 2H), 7.58 (d, 4H), 7.85 (dd, 2H), 8.03 (d, 4H), 8.22 (t, 2H), 8.52(d, 2H), 8.89 (d, 2H), 9.39 (br s, 2H), 10.16 (br s, 2H), 11.91 (br s,2H). MS (EI): m/z 458 (M⁺, 100), 354 (49.1), 250 (27.6), 221 (8.9), 130(9.4), 105 (13.6), 78 (8.6). Anal. Calcd. for C₂₈H₂₂N₆O.3.5HCl (586.12):C, H, N, Cl.

2,5-Bis[4-(cyclohexylimino)aminophenyl]furan (Compound 6d) Free base:pale yellow needles, mp 242-243° C. dec (EtOAc). Yield: 17%. ¹H NMR(DMSO-d₆): 1.18-1.90 (m, 20H), 2.14 (m, 2H), 5.71 (br s, 4NH), 6.82 (s,2H), 7.63 (d, 4H). [A 41% yield of the mono-amidine/mono-amine (freebase, yellow solid, mp 195-196° C.) was islolated by chromatography onsilica (EtOAc-MeOH, 9:1). The insoluble nature of the reaction mediumwas the likely cause of the incomplete reaction.] Dihydrochloride:tan/peach solid mp 244-248° C. dec. ¹H NMR (DMSO-d₆): 1.27 (m, 6H),1.63-1.96 (m, 14H), 2.72 (m, 2H), 7.22 (s, 2H), 7.40 (d, 4H), 7.96 (d,2H), 8.60 (br s, 2H), 9.34 (br s, 2H), 11.39 (br s, 2H). MS (FAB,thioglycerol): m/z 469.4 (MH⁺, 100). Anal. Calcd. forC₃₀H₃₈N₄O.2HCl.0.75EtOH.0.25H₂O (580.60): C, H, N.

2,5-Bis[4-(benzimidoyl)amino-2-methylphenyl]furan (Compound 6g) Freebase: yellow crystalline solid. Yield: 60%. ¹H NMR (DMSO-d₆): 2.48 (s,6H), 6.50 (br s, 4NH), 6.75 (s, 2H), 6.84 (s, 4H), 7.44 (m, 6H), 7.71(d, 2H), 7.95 (d, 4H). Hydrochloride: orange/yellow hygroscopic solid.¹H NMR (DMSO-d₆): 2.61 (s, 6H), 7.03 (s, 2H), 7.38-7.44 (m, 4H),7.63-7.68 (m, 4H), 7.75-7.80 (m, 2H), 7.94 (d, 6H). MS (EI): m/z 484(M⁺, 100), 381 (87.2), 278 (37.9), 235 (5.4), 218 (3.1), 190 (5.5), 144(11.1), 103 (32.8), 76 (9.3). Anal. Calcd. forC₃₂H₂₈N₄O.2HCl.0.5H₂O.569.39): C, H, N.

2,5-Bis[2-methyl-4-(2-quinolylimino)aminophenyl]furan (Compound 6i).Free base: orange powdery crystals, mp 168-169° C. (EtOH). Yield: 52%.¹H NMR (DMSO-d₆): 2.54 (s, 6H), 6.80 (s, 2H), 6.95 (m, 4H), 7.69 (m,2H), 7.78 (d, 2H), 7.84 (m, 2H), 8.07 (d, 2H), 8.12 (d, 2H), 8.44 (d,2H), 8.50 (d, 2H). Dihydrochloride: orange solid, mp>185° C. dec. ¹H NMR(DMSO-d₆): 2.65 (s, 6H), 7.10 (s, 2H), 7.50 (m, 4H), 7.85 (m, 2H), 8.01(m, 2H), 8.20 (d, 2H), 8.26 (d, 2H), 8.46 (d, 2H), 8.80 (d, 2H), 9.44(br s, 2H), 10.21 (br s, 2H), 11.98 (br s, 2H). MS (FAB, thioglycerol):m/z 587.2 (MH⁺, 100). Anal. Calcd. for C₃₈H₃₀N₆O.2.0HCl.1.75H₂O(691.13): C, H, N, Cl.

2,5-Bis[2-methyl-4-(5-methyl-2-pyridylimino)aminophenyl]furan (Compound6j). Free base: yellow crystalline solid, mp 156-158° C. (Et₂O/hexanes).Yield: 74%. ¹H NMR (DMSO-d₆): 2.37 (s, 6H), 2.50 (s, 6H), 6.55 (br s,4NH), 6.75 (s, 2H), 6.85 (m, 4H), 7.70-7.76 (m, 4H), 8.18 (d, 2H), 8.45(s, 2H). Hydrochloride: orange solid, mp>175° C. dec. ¹H NMR (DMSO-d₆):2.49 (s, 6H), 2.62 (s, 6H), 7.08 (s, 2H), 7.43 (d, 2H), 7.47 (s, 2H),7.85 (dd, 2H), 7.98 (d, 2H), 8.03 (d, 2H), 8.42 (d, 2H), 8.74 (s, 2H),9.29 (br s, 2H), 10.07 (br s, 2H), 11.83 (br s, 2H). MS (EI): m/z 514(M⁺, 19.2), 396 (100), 278 (34.5), 144 (8.0), 118 (33.6), 91 (13.6), 43(22.8). Anal. Calcd. for C₃₂H₃₀N₆O.3.25HCl.0.75H₂O (646.62): C, H, N,Cl.

2,5-Bis[2-methoxy-4-(2-pyridylimino)aminophenyl]furan (Compound 6k).Free base: Bright yellow crystalline solid, mp 196-197° C. (EtOAc/Et₂O).Yield: 75%. ¹H NMR (DMSO-d₆): 3.92 (s, 6H), 6.64 and 6.67 (d, 2H and s,2H, overlapping a broad NH signal), 6.89 (s, 2H), 7.55 (dd, 2H), 7.86(d, 2H), 7.95 (m, 2H), 8.32 (d, 2H), 8.63 (d, 2H). Dihydrochloride:brick orange solid, mp>180° C. dec. ¹H NMR (DMSO-d₆): 4.00 (s, 6H), 7.16(s, 2H), 7.18 (d, 2H), 7.34 (s, 2H), 7.85 (dd, 2H), 8.13 (d, 2H), 8.22(t, 2H), 8.49 (d, 2H), 8.89 (d, 2H), 9.39 (br s, 2H), 10.15 (br s, 2H),11.89 (br s, 2H). MS (EI): m/z 518 (M⁺, 100), 414 (90.0), 371 (13.3),310 (12.7), 267 (9.7), 155 (6.02), 104 (25.9), 77 (9.6), 43 (13.6).Anal. Calcd. for C₃₀H₂₆N₆O.2.0HCl2.0H₂O (627.51): C, H, N, Cl.

2,5-Bis[2-chloro-4-(2-pyridylimino)aminophenyl]furan (Compound 6I). Freebase: orange crystalline solid, mp 189-190° C. (EtOH). Yield: 25%. ¹HNMR (DMSO-d₆): 6.85 (br s, 4NH), 7.02 (dd, 2H), 7.08 (d, 2H), 7.17 (s,2H), 7.56 (m, 2H), 7.93-7.89 (m, 4H), 8.29 (d, 2H), 8.64 (m, 2H).Dihydrochloride: yellow/orange solid, mp>180° C. dec. ¹H NMR (DMSO-d₆):7.45 (s, 2H), 7.60 (d, 2H), 7.80 (s, 2H), 7.86 (dd, 2H), 8.23 (m, 4H),8.50 (d, 2H), 8.90 (d, 2H), 9.54 (br s, 2H), 10.23 (br s, 2H), 11.98 (brs, 2H). MS (EI): m/z 530, 528, 526 (M⁺, 13.2, 69.8, 100), 426, 424, 422(7.9, 48, 72.4), 322, 320, 318 (2.6, 17.4, 26.6). Anal. Calcd. forC₂₈H₂₀Cl₂N₆O.2.0HCl.1.5H₂O (627.35): C, H, N, Cl.

2,5-Bis[2,6-dimethyl-4-(2-pyridylimino)aminophenyl]furan (Compound 6m).Free base: pale yellow crystals, mp 206-207° C. (EtOH). Yield: 80%. ¹HNMR (DMSO-d₆): 2.19 (s, 12H), 6.47 (s, 2H), 6.55 (br s, 4NH), 6.69 (s,4H), 7.54 (m, 2H), 7.94 (m, 2H), 8.29 (d, 2H), 8.62 (d, 2H).Hydrochloride: fluffy yellow solid, mp>xxx° C. dec. ¹H NMR (DMSO-d₆):2.28 (s, 12H), 6.68 (s, 2H), 7.28 (s, 4H), 7.84 (m, 2H), 8.21 (t, 2H),8.48 (d, 2H), 8.88 (d, 2H), 9.37 (br s, 2H), 10.12 (br s, 2H), 11.87 (brs, 2H). MS (EI): m/z 514 (M⁺, 8.5), 410 (38.7), 306 (100), 291 (16.0),148 (45.4), 104 (56.3), 77 (31.0). Anal. Calcd. forC₃₂H₃₀N₆O.3.75HCl.0.5H₂O (660.35): C, H, N, Cl.

EXAMPLE 8 Purification of Acetamidines

The following acetamidines were purified and characterized as the HBrsalt without conversion to the free base.

2,5-Bis[4-(acetimidoyl)aminophenyl]furan Dihydrobromide (Compound 6e).Fluffy tan/orange solid, mp 307-309.5° C. dec (MeOH/EtOAc). Yield: 57%.¹H NMR (DMSO-d₆, 70° C.): 2.37 (s, 6H), 7.17 (s, 2H), 7.40 (d, 4H), 7.94(d, 4H), 8.52 (br s, 2H), 9.43 (br s, 2H), 11.13 (br s, 2H). MS (FAB,thioglycerol): m/z 333.2 (MH⁺, 100). Anal. Calcd. for C₂₀H₂₀N₄O.2.0HBr(494.23): C, H, N.

2,5-Bis[4-(acetimidoyl)amino-2-methylphenyl]furan Dihydrobromide(Compound 6f). Fluffy tan/yellow solid, mp 282.5-284° C. dec(MeOH/EtOAc). Yield: 64%. ¹H NMR (DMSO-d₆, 70° C.): 2.37 (s, 6H), 2.57(s, 6H), 6.99 (s, 2H), 7.27 (d, 2H), 7.28 (s, 2H), 7.88 (d, 2H), 8.52(br s, 2H), 9.42 (br s, 2H), 11.12 (br s, 2H). MS (FAB, thioglycerol):m/z 361.2 (MH⁺, 100). Anal. Calcd. for C₂₀H₂₀N₄O.2.0HBr.0.3MeOH(531.89): C, H, N.

EXAMPLE 9 Preparation of pyridine-2-thiocarboxamide

Adapting the general method of Taylor, a mixture of 2-cyanopyridine(7.28 g, 7.0 mmol) and thioacetamide (10.52 g, 14.0 mmol) was treatedwith 60 ml of HCl-saturated DMF, and the solution was stirred vigorouslyin an open flask on an oil bath set initially at 80° C. (the temperaturegradually rising to 95° C. over the coarse of the reaction). Taylor, E.C. et al., J. Am. Chem. Soc. 1960, 82, 2656-2657. After 80 minutes (TLCmonitoring), the resulting orange suspension was cooled, neutralizedwith con. NaOH/ice, and extracted with EtOAc. The extract was washedwith water (3×) and then concentrated to a light brown solid which wastriturated with warm water and collected. The dried product was passedover a silica gel column eluting with EtOAc:hexanes (2:1) to give, afterremoval of most of the solvent and dilution with hexanes, a yellowcrystalline solid (6.36 g, 66%), mp 136-137° C.; lit mp 137° C.

5-Methylpyridine-2-thiocarboxamide. Prepared as above from2-cyano-5-methylpyridine with a reaction time of 30 minutes. SeeMoynehan, T. M. et al., J. Chem. Soc. 1962, 2637-2658. Yield: 59%. Goldcrystals, mp 172.5-173° C. ¹H NMR (CDCl₃): 2.39 (s, 3H), 7.60 (br s,NH), 7.61 (dd, 1H), 8.31 (d, 1H), 8.57 (d, 1H), 9.42 (br s, NH).

EXAMPLE 10 Preparation of S-(2-naphthylmethyl)thioimidates

The following new S-(2-naphthylmethyl)thioimidates were preparedaccording to the literature by reaction of the appropriate thioamidewith (2-bromomethyl)naphthalene in refluxing CHCl₃ (EtOH-free) for 1.5hr. See Shearer, B. G.; et al., Tetrahedron Lett. 1997, 38, 179-182.After dilution with ether, the precipitated product was collected,rinsed with ether, and dried in vacuo.

S-(2-Naphthylmethyl)cyclohexanethioimidate•HBr. Yield: 91%. White solid,mp 192-192.5° C. ¹H NMR (DMSO-d₆): 1.14-1.32 (m, 3H), 1.45-1.53 (m, 2H),1.63 (d, 1H), 1.76 (d, 2H), 1.87 (d, 2H), 2.84 (t, 1H), 4.73 (s, 2H),7.53-7.56 (m, 3H), 7.89-7.97 (m, 3H), 8.01 (s, 1H).

S-(2-Naphthylmethyl)thiobenzimidate•HBr. Yield: 94%. White solid, mp210-212° C. dec. ¹H NMR (DMSO-d₆): 4.90 (s, 2H), 7.54-7.62 (m, 2H),7.62-7.66 (m, 3H), 7.78-7.82 (m, 1H), 7.88-7.99 (m, 5H), 8.06 (s, 1H).

S-(2-Naphthylmethyl)-2-pyridylthioimidate•HBr. Yield: 58%. White fluffysolid, mp 192° C. dec. ¹H NMR (DMSO-d₆): 4.80 (s, 2H), 7.53-7.57 (m,2H), 7.59-7.62 (dd, 1H), 7.76-7.79 (m, 1H), 7.90-7.97 (m, 3H), 8.05 (s,1H), 8.10-8.14 (m, 1H), 8.26 (d, 1H), 8.78-8.80 (m, 1H).

S-(2-Naphthylmethyl)-5-methyl-2-pyridylthioimidate•HBr. Yield: 65%.White fluffy solid, mp 190-191° C. dec. ¹H NMR (DMSO-d₆): 2.42 (s, 3H),4.79 (s, 2H), 7.53-7.57 (m, 2H), 7.59-7.62 (dd, 1H), 7.90-7.97 (m, 4H),8.05 (s, 1H), 8.17 (d, 1H), 8.64 (s, 1H).

S-(2-Naphthylmethyl)-2-quinolylthioimidate•HBr. Yield: 23%. Light tanfluffy solid, mp 184-186° C. dec. ¹H NMR (DMSO-d₆): 4.73 (s, 2H),7.53-7.55 (m, 2H), 7.62-7.64 (dd, 1H), 7.78 (t, 1H), 7.87-7.97 (m, 4H),8.07 (s, 1H), 8.12 (d, 2H), 8.28 (d, 1H), 8.66 (d, 1H).

EXAMPLE 11 Biological Testing of Inventive Compounds: Materials andMethods

Differences in thermal melting values (ΔTm) were determined and DNAsamples were prepared as previously described in Boykin, D. W. et al.,J. Med. Chem. 1998, 41, 124-129 and Francesconi, I. et al., J. Med.Chem. 1999, 42, 2260-2265.

Mycobacterium tuberculosis susceptibility testing. The compounds of thepresent invention were tested against M. tuberculosis H37Rv in BACTEC12B medium using a fluorometric broth microdilution assay, theMicroplate Alamar Blue Assay (MABA), according to Collins, L. et al.,Antimicrob. Agents Chemother. 1997, 41, 1004-1009. Compounds wereinitially assessed at 6.25 ug/ml and those effecting a reduction influorescence of at least 90% relative to untreated cultures were furtherevaluated for MIC by testing at lower concentrations. The MIC wasdefined as the lowest concentration of compound effecting a reduction of≧90% of the relative fluorescence units relative to a control culture.Antimycobacterial data were provided by the Tuberculosis AntimicrobialAcquisition and Coordinating Facility (TAACF)) through a research anddevelopment contract with the U.S. National Institute of Allergy andInfectious Diseases.

Antifungal Test organisms. The fungi used in this study for all thecompounds in Table 1 included two reference strains C. albicans A39 andAspergillus fumigatus (strain 168.95). Expanded studies on 6j employedthe fungi listed in Table 3.

Medium. Antifungal susceptibility testing was performed with RPMI 1640medium (Sigma Chemical Co., St. Louis, Mo.) with glutamine, but withoutsodium bicarbonate and buffered at pH 7.0 with 0.165 Mmorpholinopropanesulfonic acid.

Antifungal in vitro susceptibility testing. Experiments fordetermination of MICs of yeasts were performed by the brothmacrodilution method according to the recommendations of the NationalCommittee for Clinical Laboratory Standards. See National Committee forClinical Laboratory Standards. Reference method for broth dilutionsusceptibility testing of yeasts Document M27-T. Tentative standard,National Committee for Clinical Laboratory Standards, Wayne, Pa., 1995).The only difference compared to the standardized method was the choiceof drug dilutions, which ranged from 100 to 0.09 μg/ml. Briefly, thismethod specifies the use of an inoculum grown at 35° C. and adjusted toa concentration of 0.5×10³ to 2.5×10³ CFU/ml, incubation of the cultureat 35° C., and reading at 48 h for all yeasts except for C. neoformans,for which the results are interpreted at 72 h. The MIC was defined asthe culture with the lowest drug concentration in which a visualturbidity less than or equal to 80% inhibition compared to that producedby the growth control tube was observed.

The minimum fungicidal concentration (MFC) was determined by plating 100μl aliquots from tubes showing complete inhibition of growth onSabouraud agar plates. The lowest drug concentration that yielded threeor fewer colonies was recorded as the MFC.

Molds were tested by the same method but with the followingmodifications. Isolates were grown on Sabouraud dextrose agar at 30° C.,after adequate sporulation occurred (4 to 14 days); conidia wereharvested by flooding the colonies with a sterile solution of 0.85% NaCland 0.05% Tween 80 in sterile distilled water. Inocula were preparedwith a hemocytometer for counting and were then diluted with RPM1 1640medium to obtain a final inoculum size of approximately 0.5×10³ to2.5×10³ CFU/ml. The inoculum size was verified by plating an aliquot ofthe inoculum. The cultures were incubated at 30° C. for 48 to 72 h oruntil growth in the control tube was visible.

EXAMPLE 12 Results of Biological Testing

Melting temperatures were measured for the compounds 5 and 6 bound topoly dA•dT to obtain a qualitative evaluation of the DNA bindingaffinity of these drug candidates (Table 2). The difference in Tm valuesbetween the drug-DNA complexes and free DNA in solution (ΔTm) provides auseful tool to assess the interaction strength of the molecules withDNA. Since several of the compounds bound very strongly to poly dA•dT(Table 2), the interaction of these compounds with the Dickerson-Drewdodecamer d (CGCGAATTCGCG)₂ (SEQ ID NO:1), a DNA with a different andshorter AT sequence and different groove characteristics, was alsostudied. The reduced binding of the drugs to the dodecamer reflected bythe lower ΔTm values of the drug-dodecamer complexes (Table 2), allowedfor a better relative comparison of the DNA binding affinity of theseputative minor-groove binding compounds. TABLE 2 In vitro AntimicrobialActivities and DNA binding results for inventive compounds. C. albicansAspergillus fumigatus MTb DNA Affinities^(b) Comp. MIC MFC MIC MFC % MICΔTm ΔTm No. (μg/ml) (μg/ml) (μg/ml) (μg/ml) Inh^(a) (μg/ml) (AT) (oligo)5a 12.5 25 100 >100 3.13 21.6 10.8 6a Nt nt nt nt 1.56 19.6 7.5 6b 25 50nt nt 1.56 28.6 15.0 6c >100 nt nt nt ≦6.25 >28 15.4 6d >100 nt 100 >100≦6.25 26.7 12.8 6e >100 nt 100 >100 0 nd 15.9 6.0 6f >100 nt >100 nt 18nd 14.5 2.4 5b 1.04 2.08 33.4 33.4 ≦1 17.8 6.9 5c 10 100 100 100 16 15.22.8 5d 10 100 100 100 4 26.1 4.7 5e 10 10 100 100 8 5.9 0 5f Nt nt nt nt2.7 1.4 6g 3.12 6.25 nt nt 3.13 24.9 7.8 6h 10 10 10 100 ≦1 22.6 8.9 6i100 100 100 nt 7.5 1.1 6j ≦1 10 10 nt 2 24.3 10.8 6k ≦1 ≦1 ≦1 ≦1 ≦1 19.07.8 6l 100 >100 >100 nt 8 5.2 0 6m 10 >100 10 >100 5.7 1.2 DB 686 4.174.17 <1 DB 653 >100 >100 20.2 12.8^(a)% Inhibition at 6.25 μg/ml^(b)AT = poly dA•dT; oligo = d(CGCGAATTCGCG)₂^(c)nt = not tested^(d)nd = not determined

The parent diguanidino compound 5a showed a strong affinity for DNA asjudged by the ΔTm values for both poly dA•dT (21.6) and the dodecamer(10.8). These values compare well to those (25 and 11.7) for the parentamidine 2,5-bis[4-amidinophenyl]furan suggesting little difference inaffinities in this core furan structure for the amidine and guanidinecationic centers. Based on the comparison of ΔTm values for binding tothe dodecamer for the parent diguanidino 5a and for the various reversedamidine congeners 6a-6e, several interesting effects resulting fromstructural variation of the terminal groups are noted. First, reversedamidines bearing phenyl, substituted phenyl or cyclohexyl terminalgroups (6b, 6c and 6d) showed an increase in affinity over that of theparent diguanidino 5a. In a related series of diamidines, such anincrease in affinity with increasing bulk of terminal groups wasattributed to increased van der Waals interactions of the terminalgroups with the walls of the minor-groove and such is likely the case inthis system. Interestingly, the ΔTm value for the compound with aterminal 2-pyridyl group 6a is significantly lower than that for itsphenyl counterpart 6b. The lower affinity of 6a may suggest a differentbinding mode or different base pair selectivity for the two structurallyclosely related dicationic analogs. However, introduction of a methylgroup on each of the two phenyl rings of the 2,5-diarylfuran system witha terminal phenyl group, 6g, resulted in lowering of the ΔTm value toone similar to that of its pyridyl counterpart, 6h. The use of smallalkyl terminal groups, methyl groups as found in 6e and 6f, also led toa significant drop in binding affinity.

The placement of a single substituent on each of the two phenyl rings ofthe 2,5-diarylfuran framework produced striking differences in ΔTmvalues for both the diguanidine and reversed amidine series. Again,based on the comparison of ΔTm values for binding to the dodecamer forthe diguanidine series, it is noted that placement of single substituentof roughly the same size but of differing electronic properties on thephenyl rings resulted in a lowering of the ΔTm values; compare thevalues of 5a with 5b-5e. The most dramatic effect was observed for thestrongest electron-withdrawing group, the CF₃ of 5e, which reduced theΔTm value to zero.

The ΔTm values for a series of compounds with terminal 2-pyridyl groups(6a, 6h-6m) showed a different sensitivity to substituents. In thiscase, the introduction of a single substituent on each of the two phenylrings of the 2,5-diarylfuran framework caused little effect on the ΔTmvalues when the substituent was methyl (6h) or methoxy (6k). However,introduction of a chloro group (6l) resulted in significant reduction ofthe value, perhaps in part due to a pK effect. The detrimental effect ofthe chloro group on the ΔTm was much greater for the pyridyl derivative6l than for the analogous guanidine 5d, possibly due to the lowerbasicity of the reversed amidine in comparison to the guanidine. Inagreement with the results from the diguanidine series, the introductionof two methyl groups on each of the core phenyl rings (6m) alsodramatically reduced the DNA affinity. Interestingly, replacement of theterminal 2-pryidyl with a 2-quinoyl group (6I) also resulted in asignificant reduction of the ΔTm suggesting definite limits on thedimensions of the terminal group. On the other hand, the introduction ofa methyl group on the terminal pyridyl ring (6j) slightly enhanced thebinding affinity.

The antimicrobial data for these compounds are also summarized in Table2. The greatest activity amongst the diguanidino compounds was found for5a and 5b, which showed good in vitro activity against both Candidaalbicans and Mycobacterium tuberculosis. These compounds gave MIC valuesbetween 1 to 3 μg/ml against both organisms. Both compounds werefungicidal against C. albicans. The2,5-bis[alkylimino]aminophenyl]furans (6d, 6e, 6f), in general, did notshow significant antimicrobial activity, although compound 6d, with thelarger cyclohexyl group, did exhibit some activity against M.tuberculosis.

The 2,5-bis[arylimino]aminophenyl]furans can be divided into two groups:those in which the terminal group is phenyl or substituted phenyl andthose in which it is 2-pyridyl or substituted 2-pyridyl. The terminalphenyl group class of compounds did not exhibit high antifungalactivity; however, both 6b and 6g did show significant activity againstM. tuberculosis with MIC values of 0.78 and 1.56 μg/ml, respectively.Four of the compounds in the terminal pyridyl group class (6a, 6h, 6jand 6k) showed promising activity against M. tuberculosis with MICvalues ranging from 1.0 to 2.0 μg/ml. Most of the 2-pyridyl compoundsexhibited only moderate antifungal activity. As exceptions, 6j and 6kshowed activity at the MIC level of ≦1.0 μg/ml against C. albicans, and6k showed a similar level of activity against Aspergillus fumigatus.

In order to evaluate the spectrum of antifungal activity of thesecompounds, pyridyl-substituted amidines 6j and 6k were selected forstudies against other pathogenic fungi (Table 3). Compound 6j was quiteeffective against C. albicans and exhibited fungicidal activity againstseveral strains. Compound 6j was less effective versus both Aspergillusspecies. Compound 6j showed good fungicidal activity versus Rhizopusarrhizus; however, it was not very effective against the mold Fusariumsolani. Compound 6k did not show significant activity in the expandedfungus panel.

To summarize, dicationic 2,5-bis(4-guanidinophenyl)furans 5a-5f,2,5-bis[4-(arylimino)aminophenyl]furans 6a-6c, 6g-6m, and2,5-bis[4-(alkylimino)aminophenyl]furans 6d-6f have been synthesizedstarting from 2,5-bis[tri-n-butylstannyl]furan. Thermal melting studieswith poly dA•dT and the duplex oligomer d(CGCGAATTCGCG)₂ (SEQ ID NO: 1)demonstrated high DNA binding affinities for a number of the compounds.Both the guanidines and the reversed amidines in the 2,5-diarylfuranseries have strong DNA binding properties.

Compounds in both these classes show both antifungal andanti-mycobacterial activity. In addition, they may be broad-spectrumanti-fungal agents. Of the nineteen novel dicationic compoundssynthesized, six (6a, 6b, 5b, 6h, 6j, 6k) exhibited MICs of 2 μg/ml orless versus Mycobacterium tuberculosis. Of the nineteen screened againstCandida albicans, four gave MICs of 2 μg/ml or less (5a, 5b, 6j, 6k) andtwo (5a, 6k) were fungicidal, unlike a standard antifungal drugFluconazole which was fungistatic. One of the tested compounds (6k)exhibited a MIC of 1 μg/ml and was also fungicidal for Aspergillusfumigatus. Some compounds possessed inhibitory activity againstCryptococcus neoformans but all appeared less potent for this pathogenicyeast compared with C. albicans and A. fumigatus.

Evaluation against Trypanosoma brucei rhodesiense in vitro showed thatthe (arylimino)aminophenyl furans, especially when aryl is 2-pyridyl,were effective in the 0.02 to 0.1 μg/mL range; approximately 1/10 aseffective as pentamidine and furamidine. In contrast, these newcompounds are ten times more effective than pentamidine and furamidine,and comparable in activity to benznidazole, against T. cruzi. TABLE 3Evaluation of Compound 6j against an Expanded Fungus Panel Genus,species, isolate number MIC 80% MIC 100% MFC Aspergillus flavus 194.993.12 6.25 >100 Aspergillus flavus 107.96 3.12 50 >100 Aspergillus flavus141.88 3.12 50 >100 Aspergillus fumigatus 168.95 50 50 >100 Aspergillusfumigatus 182.99 3.12 50 >100 Aspergillus fumigatus 119.00 3.12 50 >100Aspergillus fumigatus 165.86 50 50 >100 Aspergillus fumigatus 153.903.12 50 >100 Fusarium solani 152.89 3.12 50 50 Rhizopus arrhizus 117.890.78 1.56 1.56 Candida albicans 116.98 1.56 1.56 >100 Candida albicans159.95 0.79 1.58 3.12 Candida albicans 149.97 0.78 1.56 6.25 Candidaalbicans 156.97 1.56 3.15 >100 Candida albicans 126.97 1.56 3.12 3.12Candida albicans 117.00 0.78 3.12 25 Candida albicans A39 1.56 1.56 25Cryptococcus neoformans H99 1.56 1.56 >100MIC and MFC values are μg/ml.MIC 80% = 80% of inoculum is inhibited.MIC 100% = 100% of inoculum is inhibited.MFC = minimum fungicidal concentration.

In the specification, and examples there have been disclosed typicalpreferred embodiments of the invention and, although specific terms areemployed, they are used in a generic and descriptive sense only and notfor the purposes of limitation, the scope of the invention being setforth in the following claims.

1. A compound according to Formula I:

wherein: R₁, R₂, R₃ and R₄ are each independently selected from thegroup consisting of H, alkyl, alkoxy, halide, and alkylhalide groups; R₅is H, alkyl or aryl;. R₆ is H, alkyl, aryl, or NR₇R₈, wherein R₇ and R₈are each independently selected from the group consisting of H, alkyland aryl; and X is O, S or NR₉, wherein R₉ is H or alkyl.
 2. Thecompound according to claim 1, wherein R₁ and R₂ are each an H.
 3. Thecompound according to claim 1, wherein R₁ and R₂ are each an H and R₃and R₄ are each lower alkyls.
 4. The compound according to claim 1,wherein R₃ and R₄ are each a halide.
 5. The compound according to claim1, wherein R₃ and R₄ are each alkoxy.
 6. The compound according to claim1, wherein R₃ and R₄ are each alkyl halides.
 7. The compound accordingto claim 1, wherein R₅ is an H, R₆ is a NR₇R₈, and R₇ and R₈ are each anH.
 8. The compound according to claim 1, wherein R₆ is a pyridyl.
 9. Thecompound according to claim 1, wherein R₆ is a substituted pyridyl. 10.The compound according to claim 1, wherein R₆ is a quinolinyl.
 11. Apharmaceutical composition comprising a compound according to Formula I:

wherein: R₁, R₂, R₃ and R₄ are each independently selected from thegroup consisting of H, alkyl, alkoxy, halide, and alkylhalide groups; R₅is H alkyl or aryl; R₆ is H, alkyl, aryl, or NR₇R₈, wherein R₇ and R₈are each independently selected from the group consisting of H, alkyland aryl; and X is O, S or NR₉, wherein R₉ is H or alky; in apharmaceutically acceptable carrier.
 12. The pharmaceutical compositionof claim 11, wherein the composition is formulated for parenteraladministration.
 13. The pharmaceutical composition of claim 11, whereinthe composition is formulated for oral administration.
 14. Thepharmaceutical composition of claim 11, wherein the composition isformulated for topical administration.
 15. A process for preparing apharmaceutical composition comprising formulating the compound of theformula (I) according to claim 1 and optionally a pharmaceuticallyutilizable carrier.
 16. A method of treating an microbial infection in asubject in need of such treatment, wherein the microbial infection iscaused by a microorganism selected from the group consisting ofMycobacterium tuberculosis, Trypanosoma spp., Candida albicans,Aspergillus spp., Cryptosporidium parvum, Giardia lamblia, Plasmodiumspp., Pneumocystis carinii, Toxoplasma gondii, Fusarium solani, andCryptococcus neoformans, said method comprising administering to thesubject a compound according to Formula I or a pharmaceuticallyacceptable salt thereof:

wherein: wherein R₁, R₂, R₃ and R₄ are each independently selected fromthe group consisting of H, alkyl, alkoxy, halide, and alkylhalidegroups; R₅ is H, alkyl or aryl; R₆ is H, alkyl, aryl, or NR₇R₈, whereinR₇ and R₈ are each independently selected from the group consisting ofH, alkyl and aryl; and X is O, S or NR₉, wherein R₉ is H or alkyl. 17.The method according to claim 16, wherein the compound is administeredparenterally.
 18. The method according to claim 16, wherein the compoundis administered orally.
 19. The method according to claim 16, whereinthe compound is administered topically.